Within eukaryotic cells, genome duplication initiates at multiple sites on each chromosome. Replication initiation events in diploid mitotic cells proceed in a precise order and are strictly regulated by a series of cell cycle checkpoint signaling pathways. These regulatory constraints, however, are often relaxed in cancer cells. Because the processes that coordinate replication ultimately converge on chromatin, understanding the molecular events that precede DNA replication at the chromatin level is crucial if we are to fully understand cell growth. Critical information about this process is missing because protein complexes that initiate chromosomal replication seem to bind DNA indiscriminately. To gain a complete understanding of the DNA replication process we must resolve how this non-specific DNA binding translates into highly coordinated replication. Our studies are based on the hypothesis that sequence-specific signaling molecules associate with replication initiation sites on chromatin where they modulate the local activity of the ubiquitous replication machinery and dictate both the location and timing of replication initiation events. To test this hypothesis, we characterize protein-DNA interactions at replication initiation sites and identify interactions that play regulatory roles in the DNA replication process. We use two approaches to characterize DNA-protein interactions at replication initiation sites. The first approach utilizes distinct DNA sequences, termed replicators, which facilitate the initiation of DNA replication. We have initially identified these replicator sequences and we now use them as bait to isolate protein complexes that potentially regulate replication. In recent studies we have identified two discrete DNA-protein complexes within one replicator element. One of these complexes includes chromatin remodeling proteins that determine both replication timing and transcriptional activity (Mol Cell Biol. 31:3472-84; 2011). Another complex includes RepID, a member of the DDB1-Cul4-associated-factor (DCAF) family, which binds a subset of replication initiation sites and is required for replication at those sites (Nat Commun. 8;7:11748; 2016). Our studies have demonstrated that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. We have characterized RepID interactions with other proteins, identified RepID protein partners using a non-biased approach and pinpointed protein domains within RepID that facilitate DNA-protein and protein-protein interactions. Our analyses demonstrate that RepID binding origins require RepID for initiation of DNA replication, providing the first example of a site-specific interaction that determines the initiation of DNA replication on a group of metazoan replication origins. The second approach involves developing tools to map replication initiation sites throughout the genome, and using these tools to analyze DNA replication in the context of chromatin modifications and transcriptional activity. The developed methods involve massively parallel sequencing and single-fiber imaging of replication fork progression. These procedures allow us to study the dynamics of DNA replication at the whole-genome level. Using this methodology we can test whether groups of replication initiation sites share specific properties - for example, if they associate with a particular chromatin feature. We can also identify groups of initiation sites that respond in a similar fashion to a cellular challenge, and test whether distinct groups of replication initiation sites are regulated through association with particular proteins (such as RepID). We have generated a comprehensive dataset of replication initiation sites for several human cancer cell lines (Genome Res. 21:1822-32, 2010; Epigenetics and Chromatin 9:18, 2016). Our recent comprehensive analysis of chromatin modifications associated with DNA replication origins (Epigenetics and Chromatin 9:18, 2016) demonstrated that replication origin usage varies with tissue type, with distinct modifications associated with cell-type specific replication origins. To facilitate these studies, we have developed a web-based tool (Coloweb; BMC Genomics 16:142. 2015) to help decipher the relationships among RepID binding sites and epigenetic features. This tool is available to the community to support bioinformatics characterization of DNA-protein interaction loci. We also use genome-wide data to identify DNA and histone modifications that associate with replication initiation events. For example, we observed strong associations between replication initiation and both DNAse hypersensitive sites and dimethylated histone H3 lysine 79, which exhibits a dynamic cell cycle distribution (PLoS Genet. 9:e1003542, 2013). We have also demonstrated (Nucleic Acids Research, in press) that a phosphorylated form of the NAD+-dependent deacetylase, SIRT1, binds potential replication origins and prevents replication from initiating in a subgroup of those potential origins (dormant origins). In collaborative studies, we have characterized an interaction between late-replicating origins and ORCA/LRWD1, a component of heterochromatin (Nucleic Acids Research 45:2490-2502). In another collaborative study, we have used phased allele-specific analyses of replication origins to decipher the sequence requirements for replication initiation (Nature Communications 6:7051. 2015). An important aspect of our work pertinent to human health is the response of the replication machinery to perturbations. A large and increasing number of anti-cancer drugs target DNA replication or interfere with cell cycle signaling. Understanding specific cell cycle defects in different cancers is likely to provide clues regarding their sensitivity to anti-cancer therapies. We are currently studying replication origins activated in response to those drugs, directly mapping chromatin targets involved in preventing excess replication. Our strategy consists of combining genome-scale sequencing with single-fiber analyses. This approach can provide important insights into the organization of replication initiation events and the cellular responses to signals that might perturb DNA replication. We ask how particular replication and repair pathways affect the pace and frequency of DNA replication. We observed that a DNA repair endonuclease, Mus81, modulates the pace of DNA replication in the absence of exogenous stress and that its presence is essential to help cells restore DNA synthesis in the presence of drugs that slow replication (Nature Communications 6:6746. 2015). In the future we will investigate how protein-DNA interactions that are required for DNA replication are modulated in response to environmental challenges and anti-cancer drugs. As we learn more about local and distal interactions that promote DNA replication, we will continue to explore pathways that signal back from chromatin to the cell cycle machinery to affect the replication landscape and modulate the response to anti-cancer therapy.